Purification of therapeutic or diagnostic biological molecules or entities, such as antibody, plasmid DNA, vaccines, or plasma fractions from endogenous impurities (host cell DNA or proteins) or adventitious contamination (endotoxin, virus or bacterium) is a growing field in bioseparation and bioprocessing. Large amounts of pure antibodies may often be necessary for immunological and therapeutical applications. In the last few years, monoclonal or recombinant antibodies and their constructs have become the largest class of molecules that are being investigated in clinical trials for therapeutics and diagnostics. Complementary to expression systems and production strategies, efficient purification protocols are required to obtain highly pure antibodies in a simple and cost-efficient manner. There is a need for developing less expensive, more scalable and faster purification techniques.
Traditional methods for purification, such as salt-based protein fractionation, or ultracentrifugation are not economical and are time consuming Bead-based chromatographic techniques, each relying on specific molecular interactions have been used for purification of antibodies and viruses as well and include affinity, hydrophobic interaction and ion-exchange chromatography. Chromatography, primarily on beads, has been used for purifying antibodies or viruses. Commonly used chromatographic techniques are based on the principle of interactions, including affinity chromatography, hydrophobic interaction chromatography, or ion-exchange chromatography. Efforts have been made in the past to design optimal stationary phases for each specific separation purpose. Such a stationary phase often comprises a support or base matrix attached to a ligand comprising binding groups. A combination of various chromatographic techniques may be used to develop a multimodal chromatographic separation technique for purification of biological macromolecules with higher purity and yield, in a cost-effective and efficient manner.
Alternatively, membrane chromatography has been used to achieve high efficiency and high-flux separations of biological molecules. However, to optimize a process related to the purification of a specific target molecule, a unique operating condition is often required. Moreover, the best separation matrix may vary for different samples, such as, antibody, endotoxin, or virus. For example, in the biotech industry, specific processes need to be designed for the purification of peptides and proteins (e.g., antibody); nucleic acids; or viruses. Moreover, for purification of antibodies, the type of antibody may be significant for the choice of separation matrix. Thus, alternative separation matrices are in need to provide a broad spectrum of choices for purification of the many new products that are constantly being developed.
Trace impurities may affect the capacity of membranes. Various membranes and chromatography resins have conventionally been used to remove trace impurities, such as DNA, host cell proteins, or protein aggregates. Proteins, sometimes form aggregates during freezing, and thawing procedures or process hold steps in downstream purification. Therefore, there is a constant need for removal of aggregated protein and trace impurities before proceeding to the viral clearance step and final formulation of the target protein molecule.